Identification of tissue for debridement

ABSTRACT

Provided are methods of determining whether a cell in a tissue site is viable or nonviable. Also provided are methods of debriding tissue from a tissue site. Further provided are kits comprising a compound that distinguishes between viable and nonviable cells and instructions for using the compound on a tissue site. Additionally, the use of a compound that distinguishes between viable and nonviable cells is provided, where the use is to determine whether a cell in a tissue site is viable or nonviable. Also provided is a use of a compound that distinguishes between viable and nonviable cells, where the use is for the manufacture of the above-described kit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Continuation patentapplication Ser. No. 13/231,251 filed Sep. 13, 2011 now U.S. Pat. No.8,221,989, which is a continuation of U.S. patent application Ser. No.12/265,566, filed Nov. 5, 2008 now U.S. Pat. No. 8,034,573, which claimsthe benefit of U.S. Provisional Application No. 61/002,107, filed Nov.5, 2007, and U.S. Provisional Application No. 61/002,547, filed Nov. 8,2007, all of which are incorporated by reference in their entirety.

BACKGROUND

1. Field of the Invention

The present invention relates generally to tissue treatment systems andin particular to methods of determining viability of cells in vivo.

2. Description of Related Art

Wounds, however created, require aggressive debridement in order tosatisfactorily remove any foreign or infectious material. Other detritusand necrotic tissue must also be removed in order to insure successfulprogression along the wound healing pathway. Early identification ofviable vs. non-viable tissue would be useful to both the surgeon and thepatient. Not only would it keep the patient from enduring additional,painful surgery, but it may also help with treatment outcomes (i.e.reducing the severity of cases, preventing removal of viable tissue andenhancing functionality). Also, identification of non-viable tissuewould provide a higher level of confidence that the correct tissue wasbeing removed and the right amount of tissue was being removed. Properidentification of tissue as non-viable would mean that one was lesslikely to leave visually marginal tissue.

In the case of burns, large traumatic wounds, and some chronic wounds,there exist multiple zones of tissue damage. For example, in traumaticmuscle injuries, injury may cause irreversible atrophy of the muscle. Insuch cases, free muscle transfers may be performed to restore somefunction. In certain cases, it may take up to a year to determine thatthe muscle is non-viable and surgery is required. It is known that earlytreatment (muscle transfer) may lead to better outcomes, and that delayin treatment limits the reconstruction options (Barrie et al. 2004). Onthe other hand, the more healthy tissue that remains followingdebridement, the better the outcome. Identification of viable tissueswould prevent inadvertent removal of viable tissue.

The differential levels of tissue damage are perhaps most classicallydescribed as the “Jackson zones” identified in burns (Jackson, 1953).The most severely and irreversibly damaged area is known as the zone ofcoagulation due to the destruction of the local proteins. This area isclearly unsalvageable. It is necrotic, often blackened and charred, andmust be removed. The most peripheral and least damaged area is known asthe zone of hyperemia. Tissue in this region generally completelyrecovers from the trauma unless it becomes infected or suffers prolongedhypoperfusion.

Tissue in the intermediate zone of stasis has been injured and ispotentially salvageable. This is known as the zone of stasis. In mid todeep burn injuries, these wounds can not be salvaged or convert to thezone of necrosis. As reported by Molnar et al. (2005), “This ischaracterized by increased vascular permeability, edema, and progressiveblood viscosity, leading to thrombosis and additional tissue death. Itis this zone of stasis that represents the deep second-degree burn thatis clearly viable tissue when the patient arrives but subsequently goeson to die and requires excision and grafting much in the manner of athird-degree or full-thickness burn.” When this occurs, wound healing isimpeded, and the patient may have to go back in for additional, painful,debridement.

As stated above, the viability of the tissue is dependent upon theability of the cells in this area to recover from the physiologicalinsults arising from the burns. If the cells are able to receiveadequate perfusion and nutrients in a timely fashion, the tissue maysurvive. If on the other hand, this does not occur, as edema increases,perfusion decreases, tissue oxygenation decreases and the injuryprogresses resulting in cell and tissue death over the 48-72 hourspost-injury. Similar zones of injury, albeit not as visually striking,occur in traumatic wounds, as well as chronic wounds such as decubitus,to various tissues.

The Faustian quandary for the surgeon is whether to (1) conservativelydebride, allowing some of the marginal tissue to stay in place andweighing the balance between whether the tissue will respond to theresuscitation efforts or whether the tissue will succumb, becomenecrotic, provide a nidus for infection and have to be removed at asubsequent procedure or (2) aggressively debride well beyond the marginof the clearly injured tissue, potentially removing viable orrecoverable tissue, and by taking this wide swath of tissue severallylimiting options for future reconstructive options and hence,functionality.

Currently, debridement and tissue removal in traumatic injuriesgenerally depends upon the surgeon's knowledge of viable tissuemorphology. However, in many instances this is not 100% accurate. Areasmay look questionable, and it is not until later that it is determinedthat the tissue is nonviable. At this point, another trip back to theoperating room, and another painful debridement is warranted.Conversely, traumatic injuries may be treated by aggressive tissueremoval. Tissue which may be viable or recoverable may be removed,limiting future functionality.

Thus, in wound healing, repeated surgical debridement procedures can berequired. If senescent or non-viable cells are left at the wound edge,the wound may fail to progress towards healing. A need therefore existsfor a method that provides clear identification of the areas of thetissue that have exhibited clear markers of having succumbed to theinjury. The surgeon will then know which areas should be excised at thetime of the debridement and which should be allowed to remain so thatthe tissue can recover from the insult and serve as a platform for anyreconstruction that may be required in the future.

Efforts to identify senescent tissue in vivo include those of US2007/0197895 A1, describing an instrument that emits and receivesacoustic signals. Also, WO07/130,423A2, describes methods foridentifying a margin for debridement by obtaining tissue samples fromthe tissue site and evaluating expression profiles of the samples, wheretissue from within a wound has a different gene expression profile fromtissue adjacent to a wound.

There is a need for additional methods that allow a precise andunambiguous continuous identification of viable and nonviable cells in atimely manner, e.g., on the edge of wounds, to determine wheredebridement should take place. The present application addresses thatneed.

SUMMARY OF THE INVENTION

Problems presented by existing methods of surgical debridement aresolved by the systems and methods of the illustrative embodimentsdescribed herein. In one embodiment, a method of determining whether acell in a tissue site is viable or nonviable is provided that comprisesadding a compound that distinguishes between viable and nonviable cellsto the tissue site, then determining whether the compound indicates thatthe cell is viable or nonviable.

In another embodiment, a method of debriding tissue from a tissue sitecomprising viable and nonviable tissue is provided that comprises addinga compound that distinguishes between viable and nonviable tissue to thetissue site, then determining where viable and nonviable tissue is inthe tissue site, then debriding the nonviable tissue surgically.

Also provided is a method of determining whether a cell in a tissue siteis viable or nonviable. The method comprises visualizing the tissueunder conditions where a viable cell can be distinguished from anonviable cell.

In an additional embodiment, a kit is provided that comprises a compoundthat distinguishes between viable and nonviable cells, and instructionsfor using the compound on a tissue site by the above-described methods.

In a further embodiment, the use of a compound that distinguishesbetween viable and nonviable cells is provided, where the use is todetermine whether a cell in a tissue site is viable or nonviable.

Also, a further use of a compound that distinguishes between viable andnonviable cells is provided, where the use is for the manufacture of theabove-described kit.

Other objects, features, and advantages of the illustrative embodimentswill become apparent with reference to the drawings and detaileddescription that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is diagrams and photographs illustrating an embodiment of theinvention.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In the following detailed description of the illustrative embodiments,reference is made to the accompanying drawings that form a part hereof.These embodiments are described in sufficient detail to enable thoseskilled in the art to practice the invention, and it is understood thatother embodiments may be utilized and that logical structural,mechanical, electrical, and chemical changes may be made withoutdeparting from the spirit or scope of the invention. To avoid detail notnecessary to enable those skilled in the art to practice the embodimentsdescribed herein, the description may omit certain information known tothose skilled in the art. The following detailed description is,therefore, not to be taken in a limiting sense, and the scope of theillustrative embodiments are defined only by the appended claims.

The inventors have developed methods for determining whether tissue isviable or nonviable in situ. These methods allow precise determinationof which cells are viable and which are nonviable in a tissue site.Because of the precision of several embodiments of these techniques inidentifying specific cells that are viable or nonviable, debridement canbe more accurate in removing nonviable cells and leaving viable cellsintact. Indeed, with the present methods, techniques that targetindividual cells or small groups of cells, such as laser dissectiontechniques, become more useful in debridement procedures becausespecific cells can be identified as viable or nonviable. Thus, withthese techniques, the physician can precisely remove more nonviabletissue and leave more viable tissue intact than with currently practiceddebridement procedures.

In some embodiments, the application is directed to a method ofdetermining whether a cell in a tissue site is viable or nonviable. Themethod comprises adding a compound that distinguishes between viable andnonviable cells to the tissue site, then determining whether thecompound indicates that the cell is viable or nonviable.

These methods and all other methods described herein can be used withany vertebrate including birds, reptiles and any mammal, includinghorses, cats, dogs, cows, sheep, goats, pigs and humans.

As used herein, a cell is viable if it is alive and not destined to die,e.g., by apoptosis, necrosis or senescence. “Viable cell” also includesa cell that is ‘stunned’ but alive, i.e., cells that sustained somedamage, e.g. cells at the margin of a burn that were heated and may notfunction properly for a time, but would be expected to recover.Generally, a cell would be expected to recover if it has not lost itsintegrity. This can be measured by determining whether the cell ispermeable to certain dyes, whether metabolites are leaking excessivelyinto the surrounding tissue, whether intracellular or intraorganelleproteins (e.g., cytochrome c) or enzymes (e.g., esterases) are presentoutside the cell, by the various methods described herein, or any otherprocedure known in the art.

The compound can be formulated in any manner known in the art. Theskilled artisan can determine, without undue experimentation, a usefulformulation for any particular application. Useful formulations includea gel, spray, liquid, powder, cream, lotion, ointment, suspension,sheet, or other solid, semisolid or liquid which can be dusted onto,painted on, sprayed into, poured on, laid over or otherwise administeredinto or onto the tissue site so that it can come into contact thereto.

As used herein, a sheet is a broad, relatively thin coherent mass orpiece of material, including those made from bioabsorbable ornonbioabsorbable materials, or both. Examples include pads, sponges,paper and thin membranes. The sheet can be transparent or opaque and canbe in any two-dimensional shape. The compound can be covalently bound orsimply adsorbed to, or absorbed into, the sheet.

Any of the compound formulations can include any other agents, includingbioactive agents, e.g., an antibiotic or a growth factor. Nonlimitingexamples of growth factors which could be useful in the compoundformulations include a vascular endothelial growth factor (VEGF), afibroblast growth factor (FGF), a platelet derived growth factor (PDGF),an angiogenin, an angiopoietin-1, a del-1, a follistatin, a granulocytecolony-stimulating factor (G-CSF), a hepatocyte growth factor/scatterfactor (HGF/SF), an interleukin-8 (IL-8), an IL-1β, an IL-1, an IL-6, aleptin, a midkine, a placental growth factor, a platelet-derivedendothelial cell growth factor (PD-ECGF), a platelet-derived growthfactor-BB (PDGF-BB), a PDGF-AB, a pleiotrophin (PTN), a progranulin, aproliferin, an epidermal growth factor (EGF), a keratinocyte growthactor (KGF), an activin A, a transforming growth factor-α (TGF-α), atransforming growth factor-β (TGF-β), a tumor necrosis factor-α (TNF-α),a vascular endothelial growth factor (VEGF), a matrix metalloproteinase(MMP), an angiopoietin 1 (ang1), an ang2, and a delta-like ligand 4(DLL4).

The compound in these methods include anything that can be used todistinguish viable from nonviable tissue. In some embodiments, thecompound identifies a molecule that has a differential expressionpattern in viable vs. nonviable cells. The differential expressionpattern could be in quantity (e.g., a different amount of the moleculeis present in, or is released by, nonviable cells vs. viable cells) orlocation (e.g., intercellular vs. extracellular presence of themolecule).

In some embodiments, the compound comprises an antibody binding site,e.g., an antibody, an Fab fragment, an F(ab)2 fragment, or aheterologous protein engineered to comprise an antibody binding site.

In other embodiments, the compound comprises an aptamer. Aptamers areoligonucleotides produced in vitro which are generally used to bind tospecific proteins, but which may also be used to bind to cells. Aptamerscan be prepared by an iterative selection process to bind specificallyand tightly to most proteins or other molecules (Brody and Gold, 2000).Because of their specificity and binding abilities, aptamers arebelieved to have great potential as diagnostic agents (Brody and Gold,2000; U.S. Pat. No. 7,052,854). Aptamer preparation does not requireeither animal or cultured cells. Aptamer synthesis may be conductedthrough PCR, and the resulting aptamers are stable at room temperatureand have a long shelf life. Visualization of the aptamer-bound proteinsmay be conducted by any one of a number of different methodologies, forexample as outlined in Seal et al., 2006 and Mir et al., 2007.

The compound can also comprise a small organic molecule, e.g., less thanabout 5000 mw, 2000 mw, 1000 mw, or 500 mw. An example is a substrate ofan enzyme (e.g., isopropyl-β-D-thiogalactopyranoside [IPTG], which isconverted from a colorless substrate to a colored product byβ-galactosidase).

In some embodiments, the compound comprises a detectable marker, e.g.,conjugated to the antibody, aptamer or small organic molecule. Thedetectable marker can be any known in the art, provided the marker canbe detected visibly or utilizing other portions of the electromagneticspectrum, with an instrument such as a camera, a dissecting microscopeor a Geiger counter, through a filter, or after subsequent processing,such as adding an enzyme substrate to achieve a colored product. As isknown in the art, a filter is particularly useful when the detectablemarker is fluorescent, where the filter is used to block out excitationwavelengths while letting emission wavelengths pass through. Nonlimitingexamples of detectable markers are enzymes, dyes (including visible andfluorescent dyes), radioactive compounds, quantum dot-containingcompositions, and metals or metal-containing compositions (includingproteins) such as ferritin or a magnetic contrast agent such as thatdescribed in WO07069040A2. The latter compounds are particularly usefulfor using optical coherence tomographic imaging to evaluate cellviability. See, e.g., U.S. Patent Application Publication US2007/0038121 A1.

The detectable marker could also be introduced secondary to theapplication of the compound. For example, the compound could be anantibody that binds to a protein expressed in nonviable tissues but notviable tissues. To determine whether the antibody is binding to theprotein, a second antibody conjugated to the detectable moiety, such asa fluorescent dye or enzyme, is added; the tissue is then washed and thedetectable moiety is visualized. Where the detectable moiety is anenzyme, an enzyme substrate that forms a colored product is added tovisualize its presence. Alternatively, if the compound is an antibodyincorporated into a sheet, and the compound binds to a protein that isreleased from nonviable cells, the sheet may be laid onto the tissuesite allowing the protein to bind, then a second antibody that binds tothe protein is added to the sheet, where the second antibody furthercomprises a detectable label, forming an antibody-protein-antibody*“sandwich”. The detectable label (*) is then visualized. Such an assaycan be performed with the sheet in situ. Alternatively, the sheet can beremoved from the tissue site and further processed to add the labeledsecond antibody and visualize the label.

In one embodiment, the compound could comprise inert, biocompatibleparticles such as carbon black or colloidal gold bound to it, or boundto a quantum dot or use up-converting phosphor technology (UPT).

The compound, or detectable label thereon, may also be light activatedto distinguish between viable and nonviable cells. See, e.g., U.S. Pat.No. 6,057,096.

In some embodiments of these methods, the compound is a dye. Includedare viability dyes, for example a dye that is more visible orfluorescent in viable cells than in nonviable cells. Nonlimitingexamples include fluorescein diacetate, trypan blue, and calcein AM. Seealso http://probesinvitrogen.com/handbook/print.1502.html.

In one aspect of these embodiments, the compound is a dye that showsreactive oxygen species concentrations. Cells which are healthy canproduce reactive oxygen species concentrations. For example,dihydrorhodamine 123 is a hydroperoxide sensitive fluorescent probe. Inviable cells it is trapped in a non-fluorescent form. However, it isconverted to the mitochondrial selective form, rhodamine 123 byhydroperoxide. Light emission from rhodamine 123 may be recorded bydigital microscopy.

In other embodiments, the dye is more visible or fluorescent innonviable cells than in viable cells. Included here are dyes that crossthe nuclear membrane of only nonviable cells. Examples are propidiumiodide and ethidium bromide.

A dye that indicates mitochondrial death can also be used as a viabilitydye. Actively metabolizing mitochondria are characterized by high innermembrane potential. This dissipates in cells about to undergo apoptosisor necrosis. Mitochondrial activity may be visualized by JC-1 dyeaccumulation. This dye exhibits a diffuse green fluorescence in thecytoplasm of dead mitochondria and is viewed as red fluorescence whenmitochondria are active.

An alternate embodiment includes incorporation of a biocompatible dyeinto a liposome covered with the proteins that bind to the compound.Once the two components are bound to each other and the tissue site iscleaned of the formulation, the dye can be released from the liposomesvia directed energy such as ultrasonic stimulation or other appropriateenergy input.

Piezoelectric crystals capable of emitting energy in an appropriateformat for eliciting visualization is also consistent with thisinvention.

The compound can identify a molecule, for example a protein (e.g.,enzyme, electron transport protein, structural protein, membraneprotein), nucleic acid (e.g., genomic or mitochondrial DNA, an RNA),carbohydrate (e.g., polysaccharide), lipid, or small molecule (e.g., ametabolite), provided the molecule has a differential expression patternin viable vs. nonviable cells. As discussed above, the differentialexpression pattern could be in quantity or location. The molecule couldalso have a different structure in viable vs. nonviable cells, e.g., aprotein may be denatured in nonviable cells but not in viable cells.

In some embodiments, the compound identifies a protein. The proteinidentified by the compound may be elevated during the process of celldeath, e.g., during apoptosis, senescence or cell necrosis. For example,the protein may be elevated inside dead or dying cells. Alternatively oradditionally, more of the protein may be released from dead or dyingcells than from viable cells. As used herein, a protein released from acell moves from inside to outside the cell. The release can be by anactive cellular process and/or by passive leaking out of the cell, e.g.,due to increased permeability of the cell or an organelle of the cell,such as mitochondria.

Where the protein identified by the compound is released from dead ordying (i.e., nonviable) cells, the presence of the compound outside thenonviable cells may be determined by placing a sheet comprising thecompound on the tissue site, where the compound is capable of binding orreacting to the released protein on the tissue site, then determiningwhether the protein is bound or has reacted to the compound. In theseembodiments, areas of the sheet comprising protein bound to the compoundare nonviable tissue. The tissue site may then be debrided adjacent toareas of the sheet indicating nonviable tissue. After assaying fornonviable tissue using the sheet of these embodiments, the sheet, or aportion thereof, can be removed, providing a ‘map’ of the viable andnonviable areas of the tissue site. Such a removed sheet can be storedin the patient's records. At least a portion of the sheet can alsoremain on the tissue site, e.g., as a guide for debridement. The sheetcan further comprise a bioactive agent, for example an antibiotic orgrowth factor. Where present, the bioactive agent could preventinfection or stimulate wound healing, particularly when the sheet isleft on the tissue site, e.g., after debridement.

The sheet of these embodiments can be made of bioabsorbable ornon-biodegradable material, or both. When the sheet is to remain on thetissue site, a bioabsorbable sheet is often preferred. Nonlimitingexamples of materials that could be used in the sheets are starch films,collagen, nitrocellulose, regenerated cellulose, a cellulose acetate,acyl substituted cellulose acetates and derivatives thereof includingethylene-vinyl acetate polymers, polyvinylidene fluoride (PVDF),collagen, polyvinyl alcohol, poly(D,L-lactide-co-glycolide),polyglycolic acid, poly-(L-lactic acid), polyanhydrides, polysaccharides(e.g. alginate), polyphosphazenes, polyacrylates, polyethyleneoxide-polypropylene glycol block copolymer, poly(caprolactone),polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, degradable polyurethanes,polyacrylates, polyhydroxyalkanoates including polyhydroxybutyrates andpolyhydroxyvalerates, polyurethanes, polystyrenes, polyvinyl chloride,polyvinyl fluoride, poly(vinylimidazole), chlorosulphonated polyolefins,polyethylene oxide, polyvinyl alcohol, Teflon®, and nylon. In someembodiments, the sheet is a starch film, apoly(D,L-lactide-co-glycolide), a polyglycolic acid, a poly-(L-lacticacid) or a polyhydroxyalkanoate. The skilled artisan could, withoutundue experimentation, determine and obtain a sheet suitable for anyparticular embodiment of the application methods.

The compound in the sheet that identifies the protein released bynonviable cells can be a protein (e.g., an antibody), a nucleic acid(e.g., an aptamer), a small organic molecule (e.g., an enzymesubstrate), a dye, or any other compound that specifically binds to theprotein, as discussed above. The compound can further be conjugated to adetectable marker (e.g., a dye, an enzyme capable of catalyzing theconversion of a colorless substrate into a colored product, aradioactive moiety, etc.), as discussed above. In some embodiments, thereleased protein is an enzyme and the compound is a substrate of theenzyme.

In some embodiments, the protein identified by the compound is elevatedin a cell undergoing apoptosis. In other embodiments, the protein iselevated during cell necrosis or senescence. As is known in the art,apoptosis is an active process characterized by cell shrinkage,generally without an inflammatory response. Apoptotic cells are usuallyidentified using an assay involving terminal deoxynucleotidyltransferase dUTP nick end labeling, or the TUNEL assay, which detectsDNA fragmenting that is characteristic of apoptosis. By contrast,necrosis is a passive process generally involving cell swelling and aninflammatory response. Senescence is characterized by giant cells thatmetabolize but do not proliferate. It is usually induced by telomereshortening, P21 expression or γ-irradiation.

Nonlimiting examples of proteins that are elevated in the cell oroutside the cell in cell death and may be detected by the compound arecytochrome c, second mitochondria-derived activator of caspases (Smac),β-galactosidase, lipofuscin, HMGB1, NF-κB, glyceraldehyde 3-phosphatedehydrogenase, a protein with an advanced glycation end product (AGE),vimentin, lamin A, creatine kinase, peroxiredoxin 1, solublegalactose-binding lectin 7, and collagen. In some embodiments, theprotein is β-galactosidase, lipofuscin, an AGE, cytochrome c, lamin A,creatine kinase, peroxiredoxin 1, soluble galactose-binding lectin 7, orcollagen.

β-galactosidase and lipofuscin are two proteins that identify senescentcells (Gerland et al., 2003). HMGB1 is a chromatin binding protein thatis associated with both apoptosis and necrosis. In apoptotic cells,HMGB1 is immobilized and does not increase the inflammatory response.However, in necrotic cells, HMGB1 is released from the nuclei in massiveamounts and may initiate further cell death and organ necrosis (Bustin,2001; Scaffidi, 2002). Increases in NF-κB may lead to muscle wasting.Additionally, elevated AGE levels in proteins can indicate cell death.See Van Herreweghe et al., 2002. AGE are particularly common in diabetesand would thus be a particularly useful target to determine cell deathin a diabetic animal. Example 1 establishes that lamin A, creatinekinase, peroxiredoxin, a galactose-binding lectin, collagen, andfilaggrin, among other proteins, are increased in dying tissue.

A structural protein such as vimentin may be the protein identified bythe compound. Vimentin becomes externalized as cells proceed towarddeath. The presence of vimentin, or other proteins that are externalizedupon cell death, would indicate a non-viable tissue.

In other embodiments, the protein identified by the compound is depletedduring cell death. Nonlimiting examples are γ-actin, biglycan,complement component 3, fibronectin 1, α₁ proteinase inhibitor, serineprotease inhibitor 2b, transferrin, apolipoprotein A-1, pregnancy-zoneprotein, and hemoglobin (both α and β chains). Muscle cell death mayoccur if γ-actin is absent. Another example is biglycan (Schaefer etal., 2003). The inventors have discovered that biglycan is depletedduring cell death. Additionally, Example 1 describes the reduction ofcomplement component 3, fibronectin 1, α₁ proteinase inhibitor, serineprotease inhibitor 2b, transferrin, apolipoprotein A-1, pregnancy-zoneprotein, and hemoglobin in dying cells.

In another embodiment, the mitochondrial membrane potential may bedetermined. Since cells that are dead or dying do not have intactmitochondrial membranes, a difference would be indicated by themitochondrial membrane potential of live, healthy cells. A compound thatis useful for this purpose is the dye JC-1, as discussed above.

In additional embodiments, the compound specifically binds to adenatured protein that is present in nonviable tissues more than inviable tissues. Nonviable cells frequently accumulate denaturedproteins. These can be detected with, e.g., an aptamer or antibody thatspecifically binds to the denatured form of a protein but not thenondenatured form. With these embodiments, the denatured form of anyprotein can be evaluated. In some embodiments, the denatured protein iscollagen, which is abundant. Antibodies to denatured collagen arecommercially available.

The compound of these methods can, in some embodiments, detect naked DNAthat has been released from a necrotic or apoptotic cell. Naked DNA canbe detected using, e.g., dyes that bind to DNA, such as ethidiumbromide.

In other embodiments, the compound identifies an organic molecule lessthan about 5000 mw, or less than about 2000 mw, or less than about 1000mw, or less than about 500 mw. The organic molecule can be, for example,a metabolite that is released from nonviable cells more than from viablecells. Nonlimiting examples of such metabolites are ATP, glucose,glycerol, NADPH and NADH. The compound can be a component of a knownassay for the metabolite, for example luciferase or luciferin for ATP;glucose oxidase, peroxidase or a peroxidase substrate for glucose;glycerol kinase, glycerol phosphate oxidase, peroxidase or a peroxidasesubstrate for glycerol; alcohol dehydrogenase, a tetrazolium dye, orphenazine methosulfate for NADH; and glucose dehydrogenase, phenazinemethosulfate, or a tetrazolium dye for NADPH. It is contemplated thatmore than one compound can be added to the tissue site, for example, ifthe metabolite is NADH, alcohol dehydrogenase, a tetrazolium dye, andphenazine methosulfate can all be added to the tissue site, in order toeffect identification of NADH on the tissue.

With these metabolites, the method can further comprise placing a solidsheet comprising the compound on the wound, where the compound binds orreacts with the organic molecule on the wound, then determining whetherthe organic molecule is bound or has reacted to the compound. In thesemethods, any areas of the sheet comprising the organic molecule bound tothe compound is nonviable tissue.

These methods can be used to determine viability of any type ofmammalian cell present in any tissue site. Nonlimiting examples includeepidermal cells, dermal cells, fibroblasts, mesenchymal stem cells,osteoblasts, chondrocytes, myocytes, adipocytes, endothelial cells,vascular smooth muscle cells and neuronal cells.

While these methods are designed to be used on wounds, they can be usedto determine tissue viability in any tissue site including normal tissueor diseased tissue (e.g., necrotizing fasciitis). The methods can alsobe used in internal organs, e.g., prior to pancreatic necrosectomy(Parekh, 2006) or nasal sinus debridement.

The tissue site evaluated by these methods can be anywhere on or in amammal where determination of cell viability in situ is desired, e.g.,where debridement is planned. Tissue types that can be utilized includeepithelium, connective tissue, muscle tissue, pancreatic tissue andneural tissue.

These methods can be used on any tissue site. In some embodiments, thetissue site is a wound. The methods can be used on any wound where thereis a possibility of the presence of nonviable tissue. The wound may befrom, e.g., a burn, disease or trauma.

In many embodiments, the nonviable tissue identified by these methodsare selectively debrided. In some of these embodiments, the nonviabletissue is debrided with a sharp instrument, ultrasound or a hydrojet, asthey are known in the art. In other embodiments, the nonviable tissue isdebrided with a laser. Due to the accuracy of these methods inidentifying individual viable and nonviable cells, debridement with alaser is useful to eliminate nonviable cells and retain viable cells.

The application is also directed to a method of debriding tissue from atissue site comprising viable and nonviable tissue. The method comprisesadding a compound that distinguishes between viable and nonviable tissueto the tissue site, then determining where viable and nonviable tissueis in the tissue site, then debriding the nonviable tissue surgically.

In some embodiments, the tissue site is a wound. The methods can be usedon any wound where there is a possibility of the presence of nonviabletissue. The wound may be from, e.g., a burn, disease or trauma.

As with the methods described above, the compound can be selected toidentify a protein elevated in cell death. Alternatively, the compoundcan be selected to identify a protein that is depleted during celldeath.

In some embodiments, the compound comprises an antibody binding site. Inother embodiments, the compound is an aptamer. In still otherembodiments, the compound is a dye.

In some of these embodiments, the nonviable tissue is debrided with asharp instrument, ultrasound or a hydrojet. In other embodiments, thenonviable tissue is debrided with a laser.

There are also various optical methods available that allow directvisualization of viable or nonviable tissue without prior addition of acompound. Thus, the application is also directed to a method ofdetermining whether a cell in a tissue site is viable or nonviable. Themethod comprises visualizing the tissue under conditions where a viablecell can be distinguished from a nonviable cell.

In some embodiments, the tissue is visualized using optical coherencetomography. Such methods are useful for determining the condition oftissue. See, e.g., Todorovic et al., 2008. In other embodiments, thetissue is visualized using interferometry. See, e.g., Schneider et al.,1997.

In additional embodiments of this method, autofluorescence is visualizedunder conditions where tissue having high levels of autofluorescence areviable and tissue having low levels of autofluorescence are nonviable.See, e.g., U.S. Pat. No. 6,174,291. In one aspect, NADH and NADPH levelsare evaluated with these embodiments, since autofluorescence of thesecompounds decline preceding apoptosis (Toms et al. 2005). Whereautofluorescence of NADH and NADPH are evaluated, autofluorescence canbe visualized at about 460 nm with excitation at about 355 nm.

In other embodiments, the method further comprises imaging the tissuesite through a multispectral or hyperspectral camera, where the cameraimages spectra distinguishing viable and nonviable cells. Multispectralscanning has been used to determine various characteristics of tissue,including viability. See U.S. Provisional Patent Application Publication2008/0192248A1 and U.S. Pat. No. 7,366,365.

Further, multispectral and hyperspectral technology are useful foridentifying molecules with spectral properties. With hyperspectral andmultispectral imaging for these methods, the imaged spectra candistinguish a molecule with spectral properties in a viable cell fromthe molecule in or released from a nonviable cell. Molecules such ashemoglobin and cytochromes have spectral properties. Thus, in someembodiments the molecule is a cytochrome or a hemoglobin, e.g.,cytochrome c. The hyperspectral technology can identify mitochondriawith reduced versus oxidized cytochrome c, or where cytochrome c hasescaped into the cytoplasm, thereby identifying apoptotic cells.

These methods can be used on any tissue site. In some embodiments, thetissue site is a wound. The methods can be used on any wound where thereis a possibility of the presence of nonviable tissue. The wound may befrom, e.g., a burn, disease or trauma.

In many embodiments, the nonviable tissue identified by these methodsare selectively debrided. In some of these embodiments, the nonviabletissue is debrided with a sharp instrument, ultrasound or a hydrojet. Inother embodiments, the nonviable tissue is debrided with a laser.

The application is also directed to a kit. The kit comprises a compoundthat distinguishes between viable and nonviable cells and instructionsfor using the compound on a tissue site by any of the methods describedabove. The methods comprise adding a compound that distinguishes betweenviable and nonviable cells to the tissue site, then determining whetherthe compound indicates that the cell is viable or nonviable.

The various methods for determining whether a cell in a tissue is viablecan be used in vitro to determine the viability of cells in tissueengineering construct, e.g., for quality control. Using these methods,unsuitable areas can be resected prior to implantation, eliminatingundesirable responses, such as inflammatory responses, to the nonviabletissue and increasing the potential for the construct's success.

Thus, in additional embodiments, the application is directed to a methodof determining the viability of a cell in a tissue engineering constructcomprising cells. The method comprises adding a compound thatdistinguishes between viable and nonviable cells to the tissueengineering construct, then determining whether the compound indicatesthat the cell is viable or nonviable.

In some aspects of this method, the compound identifies a protein thatis elevated in cell death. In other aspects, the compound identifies aprotein that is depleted in cell death. In additional aspects, thecompound identifies an organic molecule less than about 2000 mw. Infurther aspects, the compound is a dye. The various compounds for thesemethods are as described above. In some aspects, the method furthercomprises removing nonviable cells from the tissue engineeringconstruct.

In further embodiments, the application is directed to a method ofdetermining the viability of a cell in a tissue engineering constructcomprising cells. The method comprises visualizing the tissue underconditions where a viable cell can be distinguished from a nonviablecell. As with the analogous methods described above, the tissue can bevisualized, e.g., using optical coherence tomography, interferometry,visualization of autofluorescence, or a multispectral or hyperspectralcamera. In some aspects, the method further comprises removing nonviablecells from the tissue engineering construct.

The application is additionally directed to the use of a compound thatdistinguishes between viable and nonviable cells to determine whether acell in a tissue site is viable or nonviable. In some embodiments, thecell in the tissue site is debrided if it is nonviable. In furtherembodiments, the tissue site is a wound. The methods can be used on anywound where there is a possibility of the presence of nonviable tissue.The wound may be from, e.g., a burn, disease or trauma.

Further, the application is directed to the use of a compound thatdistinguishes between viable and nonviable cells for the manufacture ofa kit. The kit comprises a compound that distinguishes between viableand nonviable cells and instructions for using the compound on a tissuesite by any of the methods described above.

The application is also directed to the use of a compound thatdistinguishes between viable and nonviable cells to determine theviability of a cell in a tissue engineering construct.

It is recognized that more than one of the above methods and uses may becombined to increase the accuracy of the determination of the boundariesof viable and non-viable tissue, and/or to have an internal check on theaccuracy of each assay. For example the above methods of using acompound that identifies β-galactosidase could be combined with themultispectral or hyperspectral methods described above.

FIG. 1 provides an illustration of one embodiment of the invention. Inthis embodiment, the tissue site is sprayed with an indicator spray of apowder or liquid comprising a compound as described above (Panel A).Examples of compounds that can be applied here include viability dyes orantibodies labeled with, e.g., a fluorescent dye. The tissue site isthen rinsed to remove compound that is not bound to the tissue (PanelB). The tissue is then illuminated, e.g., with light of the dyeexcitation wavelength (Panel C). The tissue identified here as nonviableis then debrided (Panel D). An illustration of what tissue before andafter treatment with the compound could look like is provided in PanelsE and F.

Preferred embodiments are described in the following example. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the example, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the example.

Example 1 Changes in Protein Concentrations in Injured or Dying Tissue

The following experiment was conducted to identify proteins that areincreased or decreased in injured or dying cells.

Thermal injuries were produced on the backs of rats (Rattus norvegicus)as follows. The open barrel of a 3 cc syringe was placed against theskin and filled with 60° C. water. This was left in contact with theskin for 30s, at the end of which ice water was added to cool thesyringe to room temperature. Following syringe removal, silvadene creamwas topically applied to all wounds. Wounds were removed at necropsy, ateither 4 hours or 5 days.

Tissue was divided into 1 cm pieces from the zone of necrosis. Thiscorresponds to the different Jackson Zones. Therefore the pieces shouldhave been dead at the center, the piece 2 cm from the center should havebeen viable and the intermediate zone should have shown some sign ofinjury.

Tissue was extracted for proteins and labeled using stable isotopiclabeling (iTRAQ, ABI). Protein extraction was by acetone precipitationfrom up to eight tissue types with each isolate being reduced,cysteine-blocked, digested with trypsin and subsequently given a uniquelabel. Once all samples were, individually labeled, they were combinedto make one mixture with each label identifying the unique proteinsource. To clean up the sample of any impurities such as salt build-up(a common occurrence that can impact MS evaluation) and also fractionatethe mixture, strong cation-exchange (SCX) chromatography was utilized byrunning the mixed sample through HPLC utilizing a PolyLC column whichseparated the digest into fractions by differences in charge-to-massratio. The collected fractions were then utilized for spotting onto theexcitation plate prior to its introduction to the mass spectrometer.

Information based on the “time of flight” and the peptide labeling“fingerprint,” was gathered and inputted into a protein database (inthis case Mascot) for peptide identification and matching of otherfragments. This allowed for quantification of peptide levels incomparison between normal and injured tissue. Therefore the final outputwas a determination of proteins which were either up or downregulatedbetween injured and normal tissues.

Results are provided in Table 1. “Accession” refers to a Genbankaccession number. Numbers lower than 1 in the 115:113 column indicate areduced amount of the indicated protein in necrotic tissue; numbersgreater than 1 indicate an increased amount of the indicated protein innecrotic tissue. Necrotic tissue had less hemoglobin (both α2 chain andβ chain), complement component 2, pregnancy-zone protein, fibronectin 1,alpha-1-inhibitor III (α₁ proteinase inhibitor), serine proteaseinhibitor 2b, apolipoprotein A-1 and transferrin than viable tissue.Necrotic tissue also had more lamin A, creatine kinase, peroxiredoxin 1,soluble galactose binding lection, parvalbumin, collagen, and otherproteins than viable tissue.

TABLE 1 PVal Accession Name 115:113 115:113 gi|60678292 hemoglobin alpha2 chain [Rattus norvegicus] 0.4329 2.6685E−08 gi|17985949 hemoglobinbeta chain complex [Rattus norvegicus] 0.4639 0.00251685 gi|158138561complement component 3 [Rattus norvegicus] 0.5344 2.8278E−07 gi|21955142pregnancy-zone protein [Rattus norvegicus] 0.5928 1.4014E−05 gi|9506703fibronectin 1 [Rattus norvegicus] 0.5963 0.01484368 gi|83816939alpha-1-inhibitor III [Rattus norvegicus] 0.5976 0.0008084  gi|6981576serine protease inhibitor 2b [Rattus norvegicus] 0.6383 0.00603066gi|6978515 apolipoprotein A-I [Rattus norvegicus] 0.6422 0.00111637gi|61556986 transferrin [Rattus norvegicus] 0.6465 5.7019E−10gi|50355947 lamin A isoform C2 [Rattus norvegicus] 1 2828 0.04126772gi|31542401 creatine kinase, brain [Rattus norvegicus] 1.2924 0.02160082gi|16923958 peroxiredoxin 1 [Rattus norvegicus] 1.3790 0.01324064gi|62177108 hypothetical protein LOC298795 [Rattus norvegicus] 1.38330.00547971 gi|158517925 lectin, galactose binding, soluble 7 [Rattusnorvegicus] 1 3881 0.05019835 gi|109509939 PREDICTED: similar toCollagen alpha-1(VI) chain precu 1.4249 0.02081841 gi|11968064parvalbumin [Rattus norvegicus] 1.5826 9.5076E−09 gi|56711254procollagen, type III, alpha 1 [Rattus norvegicus] 1 6331 3.8886E−06gi|158711704 collagen, type 1, alpha 1 [Rattus norvegicus] 1.74771.7774E−14 gi|16758080 procollagen, type I, alpha 2 [Rattus norvegicus]1.9007 2.8456E−08 gi|109467089 PREDICTED: similar to filaggrin [Rattusnorvegicus] 1.9949 1.6553E−09

REFERENCES

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It should be apparent from the foregoing that an invention havingsignificant advantages has been provided. While the invention is shownin only a few of its forms, it is not just limited to those forms but issusceptible to various changes and modifications without departing fromthe spirit thereof.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

We claim:
 1. A method of determining the viability of a cell in a wound,the method comprising: adding a compound to the wound that identifies aprotein having an elevated or depleted level during cell death ascompared to the protein level in healthy tissue, wherein the protein issecond mitochondria-derived activator of caspases (Smac), lipofuscin,NF-κB, a protein with an advanced glycation end product (AGE), orcreatine kinase; allowing the compound to react with the protein; anddetermining whether the protein has an elevated or depleted level as anindication that the cell is nonviable.
 2. The method of claim 1, furthercomprising: placing a solid sheet comprising the compound on the wound,wherein the compound is capable of binding or reacting to the releasedprotein on the wound tissue site, then determining whether the proteinis bound or has reacted to the compound, wherein areas of the sheetcomprising protein bound to the compound are indicative of nonviabletissue.
 3. The method of claim 2, wherein the sheet is a starch film, apoly(D,L-lactide-co-glycolide), a polyglycolic acid, a poly-(L-lacticacid), or a polyhydroxyalkanoate.
 4. The method of claim 1, wherein thecompound identifies a protein that is depleted during cell death.
 5. Themethod of claim 1, wherein the compound comprises a dye capable ofreacting with said protein.
 6. The method of claim 1, wherein thecompound comprises an antibody binding site.
 7. The method of claim 1,wherein the compound comprises an aptamer.
 8. The method of claim 1,wherein the compound comprises a detectable marker.
 9. The method ofclaim 1, wherein the compound comprises an antibody or aptamerconjugated to a detectable marker.
 10. The method of claim 1, furthercomprising selectively debriding identified nonviable tissue.
 11. Amethod of determining the viability of a cell in a wound tissueengineering construct comprising cells, the method comprising: adding acompound to the wound tissue engineering construct that identifies aprotein having an elevated or depleted level during cell death ascompare to the protein level in healthy tissue, wherein the protein issecond mitochondria-derived activator of caspases (Smac), lipofuscin,NF-κB, a protein with an advanced glycation end product (AGE), orcreatine kinase; allowing the compound to react with the protein; anddetermining whether the protein has an elevated or depleted level as anindication that the cell is nonviable.
 12. A method of determining theviability of a cell in a wound, the method comprising: adding anantibody binding site to the wound that identifies a protein having anelevated or depleted level during cell death as compared to the proteinlevel in healthy tissue; allowing the compound to react with theprotein; and determining whether the protein has an elevated or depletedlevel as an indication that the cell is nonviable, wherein the proteinis creatine kinase.
 13. The method of claim 12, wherein the antibodybinding site is an antibody.
 14. The method of claim 12, wherein theantibody binding site is an Fab fragment.
 15. The method of claim 12,wherein the antibody binding site is an F(ab)2 fragment.
 16. The methodof claim 12, wherein the antibody binding site is a heterologous proteinengineered to comprise the antibody binding site.